A variety of diagnostic tests are used to assist in the treatment of patients with infections. Currently, there are four main modalities to test for the presence of bacterial infections, which are centered on a main diagnostic technology core. These four main modalities are:
1. Microscopy;
2. Serology;
3. Molecular; and
4. Culture.
Each of these modalities has strengths and weaknesses. Microscopy can detect a large number of infections; however, it often lacks specificity to identify which species or even genus to which a particular infection belongs. Serology can remotely detect the body's immune response to an infectious agent; however, this technique assumes the patient is immuno-competent and only assays a specific bacterium at a time. Molecular diagnostics, typically based on PCR methods, is highly sensitive, but it suffers a similar issue as serology, whereby it only tests for a specific organism (or sometimes only a specific strain) at a time. Culture methods are unable to detect many strains of organisms that are currently unculturable.
Many clinical microbiological identification methods rely on passing through legacy technologies. One such technology is the culture method used as a primary enrichment step. Culture depends partially on the assumption that a disease-causing organism is cultivatable. Non-culturable organisms may be entirely missed as an etiologic agent and emerging or unique organisms could easily be misidentified. Molecular identification tests rely upon the amplification of pathogen specific DNA. These tests are sensitive; however, they usually can only detect only a limited number of organisms or genetic variants. Moreover, the starting material for molecular identification typically relies on culture methods. It is fairly well accepted that the majority of bacteria are present in polymicrobial communities and cannot be cultivated.
A number of microbial detection and identification systems have been developed. New protein-based diagnostics such as MALDI TOF mass spectroscopy systems are now approved in Europe and are pending approval in the United States. These systems include the Bruker and bioMerieux systems. These systems usually require culture first, rely on a limited reductionist diagnostic approach, or have a limited throughput.
Blood stream infections (BSIs) are now the most expensive type of hospital-acquired infection (HAI). A patient's average length of hospital stay is also affected with sepsis patients staying an average of about 23.3 days. Furthermore, it is estimated that up to 40% of patients receive inadequate initial antibiotic treatment that generates its complications and considerations. Every hour that appropriate antibiotic treatment is delayed adds to a patient's mortality rate. Delaying appropriate antibiotic treatment by up to 45 hours is an independent predicting factor for mortality in patients with S. aureus infections. This is particularly compelling when culture-based microorganism identification and the susceptibility of the identified microorganism to specific antibiotics often requires between 24 to 72 hours.
Rapid microorganism identification would improve patient outcomes. Mortality can be reduced for patients, and even more so with ICU patients. Length-of-stay reductions could also be realized; studies show that length of hospital stays could be reduced by 2 days per patient or 7 days for an ICU patient with another study showing an overall reduction by 6.2 days per patient. Another study found significant cost savings per patient for pharmacy, laboratory, and bed-related costs when rapid infection-causing microorganism identification was implemented.
Some rapid diagnostic technologies include advanced MALDI TOF, single organism PCR interrogation, and the PCR platform, Biofire. Unfortunately, most of these technologies require preceding culture methods, in which case, as noted above, uncultivable organisms are missed. Second, some of these systems have sensitivity and reproducibility issues such as a relatively high error rate in the most advanced MALDI TOF systems. Furthermore, these systems can suffer from sample volume throughput issues whereby single samples or even single colony isolates are processed one at a time. Finally, these technologies usually do not achieve adequate processing turn-around times.
One approach to identify bacteria has been to clone full-length 16S rRNA genes after polymerase chain reaction (PCR) with primers that would amplify genes from a wide range of organisms. Cloned 16S rRNA genes were sequenced by the Sanger method, which requires two or three reads to cover the entire gene. Accuracy is important because sequencing errors can lead to misclassification. The cost and effort required for the Sanger method limits the extent of sampling, and studies often produced about 100 sequences per sample. This method identifies the dominant microorganisms in a sample, but analysis of less abundant microorganisms is limited.
Accordingly: methods and kits are desired that can (1) reliably identify one or more microorganisms in a time-efficient manner, and/or (2) rapidly sequence multiple regions within microorganism genes (e.g., hypervariable regions of the genes) to reliably identify one or more microorganisms that may be present.